CA2597865A1 - Compositions for therapeutic treatment of hpv related conditions and diseases - Google Patents

Abstract

The present invention relates to the compositions comprise macro-molecules carrying human papillomavirus (HPV) E7 antigen and the use of the compositions for therapeutic treatment of HPV related conditions and diseases by inducing immune responses against E7 antigen. The invention also provides methods for preparing such compositions.

Description

FIELD OF THE INVENTION

The present invention relates to compositions for using preferably as therapeutic vaccines in the treatment of human papillomavirus infection related conditions or diseases, and also to the methods for preparing such compositions.

BACKGROUND OF THE INVENTION

Human Papillomaviruses (I-IPV) are small nonenveloped DNA viruses involved in many conditions and diseases, which are generally specific either for the skin or mucosal surfaces. The skin HPV types cause warts on hands and feet, and usually warts can persist for several months or years. Such benign tumours may be distressing for the individuals concerned but tend not to be life threatening, with a few exceptions.
The mucosal HPV types infect the anogenital region and the oral cavity.
Approximately 100 different types of HPV have been characterized to date, and approximately 40 HPV types specifically infect the genital and oral mucosa.
Infection with these types of HPV may not cause any symptoms and does not always produce visible genital warts. When symptoms do develop, they usually occur 2 to 3 months after infection with the virus. Symptoms have been known to develop, however, from 3 weeks to many years after infection occurs. As such, HPV may be spread unknowingly. Infection of Mucosal HPV types are considered one of the most common sexually transmitted diseases (STDs) throughout the world with an incidence roughly twice that of herpes simplex virus infection, many have placed the lifetime likelihood of getting genital HPV to be in the range of 75-90%. While a majority of infections are asymptomatic, infection can lead to development of genital warts and cancer of anogenital tract. Cervical carcinoma, the second most widespread gynecological cancer worldwide, is associated (>99%) with the detection of mostly HPV16 and HPV18 DNA. About one percent of women worldwide are afflicted with cervical cancer, with about 500,000 new cases occurring in the world each year, which is the most common cause of death in women under the age of 50. In addition to cervical cancer, HPV are associated with a number of anal and perianal cancers (1-8, 16).

Prophylactic vaccines have been developed against HPV. The major capsid protein of HPV can be recombinantly expressed in eukaryotic cells and the expressed major capsid protein can form authentic virus-like particles (VLPs) in the expression hosts.
The purified VLPs are effective prophylactic vaccines against HPV and they are available for preventing HPV infection (9-15). However, there are no effective therapeutic compositions available to cure infected individuals. Since viruses use so much of the host-cell machinery for their replication, it has been much more difficult to develop drugs that specifically inhibit viral replication without harming the host. It is known that the immune system can play a critical role in controlling HPV
infection.
It is well known that the incidence of HPV-induced skin warts and HPV-associated diseases increases in those who are receiving immunosuppressive treatment, suggesting that in many cases virus infection is kept under control by immunological mechanisms. Further evidence for the capacity of the immune system to control infection has come from study of spontaneous wart regression. A common observation is that in some individuals with genital warts, the warts suddenly disappear.
Such regression warts have been studied histologically, revealing a substantial influx of T
lymphocytes in the lesions. Regression is believed to be mediated by the immune system. Effective immune responses to HPV infections are thought to be mainly cell-mediated since disease can persist in individuals with serum antibodies against HPV. Moreover, it is known that spontaneous regression of warts is often accompanied by Iymphocytic infiltration, itching, reddening of the affected area and other symptoms characteristic of cell-mediated immune reaction. HPV infections are also common in patients with impaired cell-mediated immunity, where persistence of viral disease suggests poor immune surveillance. Therefore the induction of effective cell-mediated immune response against protein antigens of HPV is of major importance in therapeutic vaccine strategies against HPV infection related conditions or diseases (16-25).

Although the main target for prophylactic vaccines have been focussed on the protein, which has been shown to be effective in generating both humoral and cell-mediated immune responses, due to its restricted expression, it will not be able to induce significant therapeutic effects for HPV infection related conditions or diseases (16-24). There is however evidence that the two HPV oncoproteins, E6 and E7, are ideal targets for designing therapeutic medicines. E6 and E7, are critical to the induction and maintenance of cellular transformation and are co-expressed in the majority of HPV-containing carcinomas (16-24). Thus, therapeutic compositions targeting E6 and E7 may provide the best option for controlling HPV infection related conditions or diseases. By using appropriate antigen delivery systems (or antigen carriers), E6 and E7 antigens or their epitopes can be delivered to a host to elicit a strong, long-lasting and specific cell-mediated immune response, which can potentially have the HPV related conditions or diseases cured by eliminating infected HPV, cells infected with HPV and HPV-containing carcinomas, such as cervical cancel cells.

The L1 and L2 capsid proteins of HPV have been used as delivery systems to carry HPV E6 or E7 protein antigens, for inducing cell-mediated immune response (29-30).
As infected individuals may have developed certain immunity to HPV, or as HPV
can establish latent or persistent infection in an infected individual, and by employing numerous decoy strategies, HPV can induce immune tolerance in the host (31-32).
Indeed, despite the potent immunogenicity of the HPV capsid, only half of cervical cancer patients generate capsid-specific immunoglobulin G (IgG). The pre-existing immunity or existing immune tolerance may limit the usefulness of therapeutic vaccines developed based on HPV Ll or L2 capsid proteins.

Capsid proteins from other viruses can also self-assemble into VLPs in a suitable host when recombinantly expressed in an appropriate expression system. These VLPs can also be used as antigen delivery systems (or antigen carriers) for carrying E6 or E7 antigens or their epitopes for inducing cell-mediated immune response.
However, there are still problems related to the method of purification of VLPs (26-27), and if the VLPs are from viruses with human as hosts, the therapeutic vaccines may also possibly face the problems of preexisting immunity or existing inunune tolerance.
Thus there exists a need in the art to provide therapeutic compositions which can be used to treat HPV related conditions or diseases.

SUMMARY OF THE INVENTION

Generally speaking, the present invention resides in the discovery that the macro-molecules derived from fusion capsid-chaperone protein (FCCP) can be used as antigen delivery system (or antigen carrier) for inducing a cell-mediated immune response against carried antigens (US patent application #60944780). In this invention, the macro-molecules are used as a delivery system or vehicle to carry HPV
antigen for inducing immune responses, particularly a cell-mediated immune response against HPV antigen. Thus, the present invention relates to compositions comprising macro-molecules carrying HPV antigen. In one embodiment, the compositions can be prepared from single fusion protein, the HPV antigen and FCCP can be joined at the nucleotide level permitting expression and purification of a single fusion protein containing both HPV antigen and FCCP sequences. The separation and purification process is conducted in one or more steps in denatured condition using high concentration of chaotropic agents, such as urea or guanidine hydrochloride (Gu.HCI) solution or buffered solution. The denatured FCCP-HPV antigen fusion protein is subjected to refolding and self-assembling process to form macro-molecules containing multi-units of FCCP-HPV antigen fusion protein by a process involving gradually removing out chaotropic agents presented in the denatured FCCP-HPV
antigen fusion protein sample. As the chaperone protein in the FCCP is to facilitate the capsid protein refolding and reassembling into macro-molecules after the purification process involving using protein denaturants, such as urea and Gu.HCI, it might be desirable in some situations to have the chaperone protein clipped off by an enzymatic method and removed from the macro-molecules after refolding and reassembling process. In another embodiment, the HPV antigen can also be chemically linked or conjugated to the macro-molecules by well known methods, or HPV antigen can also join macro-molecules noncovalently. Any of several known high-affinity interactions can be adapted to nonconvalently connect HPV
anrigen with macro-molecules. In particular embodiments, the immunostimulatory substances, preferable unmethylated CpG-DNA or double strand RNA can be packaged into macro-molecules. The compositions in this invention may be used for therapeutic treatment of HPV related conditions or diseases by inducing immune responses, particularly a cell-mediated immune response against HPV antigen.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions that can be used to induce a cell-mediated immune response against HPV antigen, therefore the compositions may be used therapeutically to treat HPV related conditions or diseases. The method of preparation the compositions in this invention and the use of such compositions reside in the discovery that the macro-molecules derived from fusion capsid-chaperone protein (FCCP) can be used as antigen delivery system (or antigen carrier) for inducing a cell-mediated immune response against carried antigen. (US patent application #60944780). In this invention, the method of preparation of the compositions comprise macro-molecules carrying HPV antigen and the use of the compositions for therapeutic treatment of HPV infection related conditions and diseases are disclosed. The HPV antigen in the compositions of this invention can be any HPV gene-encoded polypeptide. Two HPV oncogenic proteins, E6 and E7, are critical to the induction and maintenance of cellular transformation and are co-expressed in the majority of HPV-containing carcinomas such as cervical cancel cells and cells within warts (17-24). Thus, it is highly desirable to use E6, E7 or both proteins in the compositions of this invention. In addition, Those skilled in the art will recognize and appreciate that in the compositions of this invention, the said HPV
antigen can be a portion of an E6 or E7 protein, provided that portion, when joined to macro-molecules (being carried by macro-molecules), retains the ability to induce a cell-mediated immune response against E6 or E7 antigen. Compositions comprise various portions of E6 or E7 rather than a complete HPV protein antigen can be produced by routine methods by those skilled in the art, such as gene-encoding E6 or E7 antigen can be deleted part of its sequence or chemically synthesized only part of its sequence, then in an expression system, recombinantly express altered DNA
sequence to produce only portion of the E6 or E7 polypeptide. In the compositions of this invention, E6 and E7 polypeptides also can be combined, such as it can be the combination of E6 and E7 proteins. Those skilled in the art will recognize and appreciate that the combination of the E6 and E7 polypeptides can be achieved by recombinantly expressing a fusion protein, or by chemically conjugating polypeptides together, furthermore, E6 or E7 polypeptide can be chemically conjugated to macro-molecules separately. In addition, in the compositions of this invention, it can be the combination of a portion of E6 and a portion of E7 proteins, provided the portion, when joined with macro-molecules, retains the ability to induce a cell-mediated immune response against E6 or E7 antigens. Each composition have a particular portion of HPV antigen or epitopes can be tested and assayed for the degree and quality of the immune responses, particularly cell-mediated immune response against HPV antigen by well known methods, such as by in vitro assay of CTL
activity, and by animal experiments described in the examples hereinafter.

The compositions of this invention can be prepared using following steps.

1. A nucleic acid which encodes FCCP and desired HPV antigen described above is incorporated into a suitable vector system as a single open reading frame with any accessory sequences proper for its expression in a chosen system. Preferably, HPV antigen is linked to the capsid protein of the FCCP as a single fusion protein.
The host cell is transformed with the vector. Transformed host cells are then cultured and the desired fusion protein is recombinantly expressed;

2. the separation and purification process is conducted in high concentration of chaotropic agents such as urea or Gu.HC1 in one or more steps; the concentration of urea or Gu.HCI solution used in the purification process can be up to 10M
urea or Gu.HCI, preferably from 4M-8M for urea and 3-6M for Gu.HCI;

3. the purified homogenous FCCP-HPV antigen fusion protein will be refolded with a process involving gradually removing out chaotropic agents presented in the purified sample by dialysis;

4. in the refolding process, the fusion protein of FCCP-HPV antigen will self-assemble into macro-molecules containing multi-units of FCCP-HPV
antigen.
The capsid protein in the FCCP-HPV antigen fusion protein can be utilized to package nucleic acids. Some nucleic acids such as double strand RNA or unmethylated CpG-DNA are well known for their ability to greatly enhance the inunune responses.
Thus it is highly desirable to have double strand RNA or unmethylated CpG-DNA
be packaged into the macro-molecules containing multi-units of FCCP-HPV antigen by adding desired double strand RNA or unmethylated CpG-DNA into purified and denatured fusion protein of FCCP-HPV antigen, then reassembling FCCP-HPV
antigen into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample.

In the FCCP fusion protein, the chaperone protein is to facilitate the refolding of the denatured capsid protein, in some situations, it may be desirable to have the chaperone protein being removed after refolding and reassembling process. The composition can be prepared by another method to have the chaperone protein being removed from the final preparation using following steps.

1. Designing a fusion protein with the capsid protein linked to HPV antigen and a chaperone protein via peptide bonds, designing an unique enzyme cleavage site at the joint of the capsid protein and the chaperone protein, the unique enzyme cleavage site is a thrombin cleavage site or an enterokinase cleavage site, or any other unique enzyme cleavage site;
2. recombinantly producing the said fusion protein containing a specific enzyme cleavage site at the joint of the capsid protein and the chaperone protein by an expression system;
3. separation and purification of the recombinantly expressed fusion protein in one or more steps in denatured form involving using high concentration of chaotropic agents, such as using up to 10M urea or Gu.HCI solutions or buffered solutions in the separation and purification process, preferably 2-8M for urea and 1-6M for Gu.HCI;
4. refolding and reassembling of the said fusion protein into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured fusion protein sample;

5. clipping off the chaperone protein from the macro-molecules by using the desired enzyme, such as using thrombin or enterokinase;

6. separating macro-molecules from the clipped off chaperone protein;

7. the final macro-molecules comprising many subunits of mostly capsid protein fused to HPV antigen.

The compositions of this invention can also be prepared using another different approach. The desired HPV antigen, for example, from E6 or E7, or both E6 and E7, or portion of E6 and E7 can be recombinantly expressed and then purified or chemically synthezed; the macro-molecules derived from FCCP can prepared according to the US patent application # 60944780. The prepared HPV antigen and macro-molecules can be conjugated or joined chemically using known techniques, such as, standard techniques involving covalent attachment for example to exposed tyrosine residues or to the epsilon-amino groups of lysine residues or the carboxyl groups of aspartate and glutamate residues. The purified HPV antigen and macro-molecules can also be joined noncovalently by affinity interactions. Any of several known high-affinity interactions can be adapted to nonconvalently connect HPV antigen with macro-molecules. For examples, a biotin group can be added to macro-molecules, the HPV antigen can be expressed as an avidin-HPV antigen fusion protein. The avidin-HPV antigen fusion protein will strongly bind the biotinlated macro-molecules.

s The capsid protein in the FCCP can be utilized to package nucleic acids. Some nucleic acids such as double strand RNA or unmethylated CpG-DNA are well known for their ability to greatly enhance the immune responses. Thus it is highly desirable to have double strand RNA or unmethylated CpG-DNA be packaged into the macro-molecules carrying FCCP-HPV antigen.
The compositions comprising macro-molecules carrying HPV antigen described herein can be used to induce enhanced immune responses, particularly cell-mediated immune response, against HPV antigen, which is capable of mediating the regression of chronic HPV infection resulted conditions or diseases, these conditions and diseases include, but are not limited to: genital warts (especially where the compositions and the infections are based on HPV of types 6 and/or 11) or cervical intra-epithelial neoplasia (especially where the compositions and the infections are based on HPV of types 16 and/or 18) in infected patients. Thus the invention provides compositions comprising macro-molecules carrying HPV antigen, which can be used for therapeutically treating HPV infection associated conditions or diseases.
The therapeutic treatment comprises administering to a patient an effective amount of compositions as described herein.

Cross-reactivity between HPV of different types has been observed, and according to such observable cross-reactivity the compositions produced hereby can be used in eliciting useful immune responses against papillomavirus types other than the types from which they were derived.

The compositions of this invention described above, are preferably used for therapeutic purpose for HPV related conditions or diseases, however, the compositions may be used for prophylactic purpose. The compositions of this invention are suitable for injection and routes and procedures of administration include, but are not limited to standard intramuscular, subcutaneous, intradermal, intravenous, oral or rectal routes and procedures. In addition, the compositions of this invention can contain and be administered together with other pharmacologically acceptable components. The compositions of this invention can also be formulated by combining with an adjuvant or other accessory substance such as an immunostimulatory molecule in order to enhance its effect as a therapeutic vaccine, and also to stimulate a preferred type of immune response in the recipient host. Useful adjuvant include, but are not limited to: double strand RNA, unmethylated CpG-DNA, aluminum hydroxide. Such adjuvant and/or other accessory substances can be used separately or in combinations as desired.

The amount of the compositions in this invention used for therapeutic or prophylactic purposes is an amount which can induce effective immune responses in a subject when administered. In addition, the amount of the compositions administered to the subject will vary depending on a variety of factors, including but not limiting to: the formulation of the compositions, adjuvant and its amount, the size, age, body weight, sex, general health and immunological responses of the subject. Effective amounts can be determined in subjects and adjustment and manipulation of established dose range are well within the ability of those skilled in the art. For example, the effective amount of compositions can be from 10 micrograms to about 1 gram, preferably from 50 micrograms to 50 milligrams. One or more doses of the vaccine may be administered at intervals. This regime can readily be optimized in subjects by those skilled in the art.

The use of the macro-molecules derived from FCCP instead of the VLPs derived from capsid proteins as HPV antigen carrier to produce the compositions described above has the following advantages:

1. FCCP or FCCP-HPV antigen fusion protein can be purified in denatured forms using high concentration of chaotropic agents such as urea or Gu.HCI. The concentration of urea or Gu.HCI solution used in the purification process can be up to lOM, preferably up to 8M for urea and 6M for Gu.HCI. The purified and denatured homogenous FCCP or FCCP-HPV antigen fusion protein subsequently can be refolded and reassembled into macro-molecules by a process involving gradually removing chaotropic agents out from the denatured FCCP or FCCP-HPV antigen fusion protein sample. Denatured capsid proteins normally cannot be correctly refolded and normally form aggregates instead of VLP
(26-27).
2. The macro-molecules formed by the self-assembling of FCCP or FCCP-HPV
antigen fusion protein may be morphologically different from authentic VLP.
The peptides displayed or exposed by authentic VLPs would be the same as the viruses, but because of the differences of the morphology, the macro-molecules may display or expose a different set of epitope peptides compare to authentic VLPs, and may have the following advantages over authentic VLPs: (1) pre-existing immunity to authentic VLPs might be circumvented by the use of macro-molecules with different morphology; (2) existing immune tolerance to authentic capsid proteins might be circumvented by the use of the macro-molecules with different morphology; (3) the use of macro-molecules with different morphology might circumvent the problem of interference with commercial anti-capsid protein assays.
3. After the purification, refolding and reassembling process, the chaperone protein might be cliped off from FCCP with a chemical method, or by an enzyemtic method, such as, a specific enzyme cleavage site can be designed at the joint of the capsid protein and chaperone protein. For example, asp asp asp asp lys can be recognized by enterokinase, and this sequence can be introduced into the joint of the capsid protein and chaperone protein, after refolding and reassembling, the enterokinase can be used to clip off the chaperone protein.

The following examples are provided in order to demonstrate and further illustrate the present invention, and are not to be construed as limiting the scope thereof.
EXAMPLES

Example 1 A chaperone protein-Hsp65 derived from Mycobacterium bovis BCG hsp65 gene (35-36) is fused to the C-terminal of the capsid protein (nucleocapsid protein or core antigen) of Hepatitis B virus (HBV) subtype ADW2 (37-38) to form a FCCP
molecule.
An E7 protein from human papillomavirus type 16 (39) is fused to the N-terminal of the FCCP molecule. The single fusion protein starting from N-terminal is E7 protein, the C-terminal of E7 protein is fused to the N-terminal of capsid protein, the C-terminal of capsid protein is fused to the N-terminal of Hsp65 protein, and can be represented as E7-Core-Hsp65. The 2479 bp of DNA sequence encoding E7-Core-Hsp65 fusion protein was chemically synthesized according to DNA
sequences from GenBank (36, 38-39).

The synthesized DNA sequence was named Ankegens 2479bp and cloned into Smal digested pBluescript II SK (+/-) from Stratagene (40) to produce pB S K-Ankeg ens -2479bp .

Example 2 Expression and purification of E7-Core-Hsp65 fusion protein E7-Core-BCG65 DNA fragment was cut from pBSK-Ankegens-2479bp by Ndel and EcoRI then subcloned into pET-23a (41) corresponding sites to produce pET-23a-2479. The pET-23a-2479 was transformed into E. coli cell line Rosetta-gami(DE3) from Novagen. E7-Core-BCG65 fusion protein was expressed in E. coli cells by fermentation and induction of transformed Rosetta-gami(DE3) cells with 0.5mM isopropyl-thio-galatopyranoside according to Novagen's pET System Manual. After fermentation, cells were harvested by centrifugation. Cells were washed once by suspending 100g cell paste in 1000m1 of buffer A(100mM Tris-Hcl pH 9.0; 5mM EDTA) then centrifuging at 8500rpm for 30 minutes. Discarded the supernatant then re-suspended the pelleted cells with 1000m1 of buffer B (50mM
sodium acetate; 2mM EDTA). The suspended cells were ruptured by homogenization process with pressure at 760bar, and then centrifuged at 8500rpm for 30 minutes. The supernatant was collected and the volume was measured. Urea was added to the supernatant according to 0.7g urea for lmi supernatant, and then sodium chloride was added to final concentration at 100 mM, L-Cysteine was added to final concentration at 20mM. The solution was stirred at room temperature to have all the urea dissolved then stirred at 4 C for overnight. After overnight stirring, the sample was applied to an XK-50 column (GE Health) containing 300ml of SP-Sepharose resin, which was previously washed with 1 M sodium chloride and equilibrated with buffer C
(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine). After sample loading, the column was washed with 10 column-volumes of buffer D(50mM
sodium acetate; 100mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine; 2.5%

Triton-X-100) overnight to remove endotoxin. After overnight washing with buffer D, the column was washed with 5 column-volumes of buffer C to remove Triton-X-100, and then the column was washed with 3 column-volumes of buffer E(50mM
sodium acetate; 300mM NaCI; 2mM EDTA; 8M urea; 10mM L-Cysteine) to remove contaminating proteins and other contaminations. E7-Core-BCG65 fusion protein was eluted from the column with buffer D(50mM sodium acetate; 800mM NaCI; 2mM
EDTA; 8M urea; 10mM L-Cysteine). Pooled eluted protein was dialyzed against 4X
40 volumes of buffer F (50mM sodium acetate, 6Murea) to remove NaC1 and L-Cysteine. After dialysis, Oxidative sulfitolysis was performed by adding sodium sulfite and sodium tetrathionate to final concentrations of 200mM and 50mM
respectively and incubating for overnight at room temperature. The sulfitolyzed sample was diluted 5 volumes with buffer F then applied to an XK-50 column with 150m1 of Q-Sepharose resin, which was previously washed with 1M NaCt and equilibrated with buffer F. After sample loading, the column was washed with 2 column-volumes of 95% buffer F and 5% buffer G (50mM sodium acetate; 1M NaCl;
6Murea), and then E7-Core-BCG65 fusion protein was eluted with a lineal gradient from 95% buffer F and 5% buffer G to 50% buffer F and 50% buffer G over 8 column-volumes. Eluted E7-Core-BCG65 fusion protein was pooled, and then dialyzed against 1X40 volumes of Tris.HCI pH9.0, 1X40 volumes of Tris.HCl pH7.5 with 100mM NaCI to remove urea and refold E7-Core-BCG65 fusion protein. The endotoxin level in the final preparation (E7-Core-BCG65 in Tris.HCl pH7.5 with 100mM NaCI) was below 5EU/mg protein.

Example 3 Therapeutic and prophylactic effects of E7-Core-BCG65 treatment in mice The E7-Core-BCG65 is an FCCP carrying an HPV16 E7 protein (antigen), and the expressing TC-1 tumor cells were used to evaluate the therapeutic and prophylactic applications of E7-Core-BCG65 on mice bearing TC-1 tumor or being challenged with TC- 1 tumor.

Female C57BL/6 mice, six to eight weeks old (20.0 2.0g) were purchased from SHANGHAI SLAC LABORATORY ANIMAL CO. LTD. Quality Control No.:
S CXK(Shanghai)2003 -0003 .

TC-1 cell line expressing HPV16 E7 protein was derived from primary lung cells of C57BL/6 mice by immortalization and transformation with HPV16 E7 gene and an activated human C-Ha-ras gene as described in Lin et al. (42).TC-1 cells were grown in RPMI1640 medium supplemented with 10% fetal calf serum, 2mM nonessential amino acids, 2mM L-glutamine, 1mM pyruvate, Penicillin/Streptomycin, and the cells were harvested by trypsinization, the cells were washed three times with PBS
then re-suspended in PBS. 1X105 TC-1 cells were inoculated subcutaneously into the mice and the mice were treated with E7-Core-BCG65 or saline subcutaneously according to their experiment groups.

Animal experiment groups:

Groups Mice Dose Time of E7-Core-BCG65 Treatment Treatment Therapeutic 8 500ug 48h and 16 days after inoculation of TC-1 Application E7-Core-BCG6 8 100ug 48h and 16 days after inoculation of TC-1 E7-Core-BCG6 8 20ug 48h and 16 days after inoculation of TC-1 E7-Core-BCG6 Prophylactic 8 100ug Two treatments with 14 days in between Application E7-Core-BCG6 Inoculation of TC-1 14 days after second 5 treatment 8 20ug 'ltvo treatments with 14 days in between E7-Core-BCG6 Inoculation of TC-1 14 days after second 5 treatment Control 6 Saline 48h and 16 days after inoculation of TC-1 The mice were monitored for the presence or absence of tumor by palpation and the volume of the tumor was measured with Vernier Caliber by 2 orthogonal dimensions twice a week; these measurements were extrapolated to mm3 and are presented as average tumor volume standard error of the mean. The life span of the mice was recorded.

In control group, the presence of the tumor was observed 4 days after TC-1 inoculation; the average volume of the tumor was grown to 40mm3 10 day after inoculation and 7499.84mm3 36 days after inoculation. All mice in the control group died within 60 days after inoculation.

In therapeutic group, mice were treated with E7-Core-BCG65 48h and 16 days after TC-1 inoculation; the average volume of the tumor was grown to 181.89mm3 (500ug), 671.34mm3 (100ug) and 2148.57mm3 (20ug) 36 days after inoculation. All mice were alive 60 days after inoculation.

In the prophylactic group, mice were treated with E7-Core-BCG65 twice in 14 days, and after second treatment, mice were inoculated with TC-1; the average volume of the tumor was grown to22.43mm3 (100ug) and 89.08mm3 (20ug) 36 days after inoculation. All mice were alive 60 days after inoculation.

Table 1 The average tumor volume in different experiment groups (mm3) (x s) Date Therapeutic Group Prophylactic Group Control (day) 500ug 100ug 20ug 100ug 20ug Group (n=8) (n=8) (n=8) (n=8) (n=8) (n=6) 8.65 5.40 16.33 8.83 42.54 24.55 * 2.32 1.06 5.56 2.91 39.00 19.28 13 41.70 20.90 51.01 20.37 84.72 36.72 * 1,97 2.44 10.58f25.56 133.57 69.64 16 31.91 12.26 49.96 20.62 189.07i91.07* 1.97 1.42 5.24 2.08 320.20 149.14 19 35.69 10.52 156.28 46.49 208.49 85.46 2.85 1,49 25.65 10.43 782.65 257.69 22 43.89 21.13 224.71 107.46 357.47 159.47 2.44 1.98 35.24 80.41 1033.81 594.12 25 109.04 47.41 257.01 107.19 756.40f258.40 4.52 2.78 17.38 6.76 2414.19 1201.87 28 127.68 56.24 395.56 128.72 892.54 364.47 18.81 5.42 69.63 24.46 4432.67 1824.46 31 156.12 49.46 525.81 152.94 1527.21 510.46 20.57 10.46 85.57t25.85 6024.54 2465.46 34 181.89 75.53 671.34 301.28 2048.57 1050.57 22.42 18.60 89.08 43.09 7499.84 3722.56 37 250.52 120.59 785.69 268.85 3051.65 1253.32 56.83 25.56 173.59 89.41 9483.58 4565.74 40 396.88 208.12 921.87 368.03 3887.08 1889.08 59.81 26.79 173.92 86.39 13141.43 5077.39 Those skilled in the art will recognize, or be able to ascertain that the basic construction in this invention can be altered to provide other embodiments which utilize the process of this invention. Therefore, it will be appreciated that the scope of this invention is to be defmed by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.

Claims (30)

1. An immunogenic composition comprising macro-molecules carrying HPV antigen, the composition induces immune responses, particularly cell-mediated immune response to the HPV antigen in a mammal to whom the composition is administered.

2. The HPV antigen of claim 1, wherein the HPV antigen is a full-length polypeptide encoded by any HPV gene or an antigenic portion thereof.

3. The HPV antigen of claim 1, wherein the HPV antigen is E6, E7 antigen or an antigenic portion thereof.

4. The HPV antigen of claim 1, wherein the HPV antigen is the combination of and E7 antigen, or the combination of the antigenic portions thereof.

5. The macro-molecules of claim 1 comprising a virus capsid protein (or nucleocapsid protein) joined via a peptide bond to a chaperone protein (or peptide), wherein the fusion protein (FCCP) can be refolded and reassembled into macro-molecules after being denatured with high concentration of chaotropic agents, such as urea or Gu.HCl concentration up to 10M, After refolding and reassembling, the chaperone protein can be clipped off and removed from reassembled macro-molecules by a chemical method or an enzymatic method.

6. The FCCP of claim 5, wherein the capsid is the protein shell of a virus. It consists of one or several monomeric subunits made of protein.

7. The FCCP of claim 5, wherein the capsid protein is a whole protein, part of the whole protein, science mutated or variant of capsid proteins which still retain the ability of self-assembly.

8. The FCCP of claim 5, wherein the capsid protein is HBV core antigen.

9. The FCCP of claim 5, wherein the chaperone protein or peptide is a member of one of the chaperone families.

10. The FCCP of claim 5, wherein the chaperone protein (or peptide) is a full length protein, a functional equivalent, such as, a fragment of whole chaperone protein, a science mutated or a variant of chaperone protein.

11. The FCCP of claim 5, wherein the chaperone protein (or peptide) is a protein (or peptide) when joined to a capsid protein (or peptide) via peptide bond, wherein the fusion protein gains the feature of being able to refold and reassemble into macro-molecules after subjecting to the treatment of high concentration of chaotropic agents such as urea or Gu.HCl. FCCP can be refolded and reassembled into macro-molecules after being processed up to 10M urea or Gu.HCl solution or buffered solution by a process involving gradually removing out chaotropic agents presented in the denatured FCCP sample.

12. The FCCP of claim 5, wherein the chaperone protein or peptide is an M.
bovis BCG hsp65 protein.

13. The composition of claim 1, wherein said HPV antigen is fused to said FCCP
by one covalent bond, wherein the covalent bond is a peptide bond.

14. The composition of claim 1, wherein said HPV antigen is bound to said macro-molecules by at least one covalent bond, wherein the covalent bond is a chemically formed non-peptide bond.

15. A method of preparation of the composition of claim 1, comprising the steps of:
A. recombinantly producing the FCCP and HPV antigen fusion protein (FCCP-HPV antigen) by an expression system;
B. separation and purification of the recombinantly expressed FCCP-HPV
antigen in one or more steps in denatured conditions involving using high concentration of chaotropic agents;
C. refolding and assembling of FCCP-HPV antigen into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured FCCP-HPV antigen sample;
D. the final macro-molecules comprising multi-units of the FCCP-HPV antigen.

16. The second method of preparation of the composition of claim 1, comprising the steps of:
A. designing an unique enzyme cleavage site at the joint of the capsid protein(peptide) and chaperone protein(peptide), such as a thrombin cleavage site or enterokinase cleavage site;
B. recombinantly producing the FCCP-HPV antigen with HPV antigen linked to capsid protein (peptide) and specific enzyme cleavage site at the joint of capsid protein(peptide) and chaperone protein(peptide), by an expression system;
C. separation and purification of the recombinantly expressed FCCP-HPV
antigen in denatured conditions in one or more steps involving using high concentration of chaotropic agents;
D. refolding and reassembling of FCCP-HPV antigen into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured FCCP sample;
E. clipping off the chaperone protein from the macro-molecules by the intended enzyme such as thrombin or enterokinase;
F. separating macro-molecules from the clipped off chaperone protein (peptide);
G. the final macro-molecules comprising multi-units of the capsid protein-HPV
antigen fusion protein.

17. The third method of preparation of the composition of claim 1, comprising the steps of:
A. recombinantly producing the FCCP and HPV antigen of claim 1 by expression systems separately, or for short HPV peptide antigen, chemically synthezing it;
B. separation and purification of the recombinantly expressed FCCP and HPV
antigen in one or more steps involving using high concentration of chaotropic agents;
C. refolding and reassembling of FCCP into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured FCCP sample;
D. conjugating or chemically linking HPV antigen with macro-molecules to form the composition of claim 1;
E. or noncovalently connecting HPV antigen with macro-molecules to form the composition of claim 1 by any of several known high-affinity interactions;
F. a method of claim 16 can be used to generate compositions with chaperone protein clipped off.

18. A method of treating or preventing HPV infection related conditions or diseases in a mammal, the method comprising administering the composition in claim 1 to the mammal in an amount effective to induce immune responses against HPV antigen.

19. The method of claim 18, wherein the composition is administered in combination with pharmacologically acceptable components, such as, carrier, diluent or vehicle.

20. The method of 18, wherein the immune responses comprise a cell-mediated immune response.

21. An immunogenic composition comprising composition of claim 1 and an immunological adjuvant.

22. The adjuvant of claim 21 is double strand RNA or unmethylated CpG-DNA.

23. The double strand RNA or unmethylated CpG-DNA in claim 22 is mixed with, or preferably packaged into the composition of claim 1.

24. A method of having the double strand RNA or unmethylated CpG-DNA in claim 23 packaged into the composition in claim 1 comprises: (a) removing contaminated nucleic acids from the FCCP or FCCP-HPV antigen fusion protein by purification process using solutions with high concentration of chaotropic agents, preferably in 4-8M urea solutions or buffered solutions; (b) adding said double strand RNA or unmethylated CpG-DNA into denatured FCCP or denatured FCCP-HPV antigen fusion protein; (c) reassembling said FCCP or FCCP-HPV
antigen into macro-molecules by a process involving gradually removing out chaotropic agents presented in the denatured FCCP or denatured FCCP-HPV
antigen fusion protein sample.

25. A method of treating or preventing HPV infection related conditions or diseases in a mammal, the method comprising administering the composition in claim 21 to the mammal in an effective amount to induce immune responses against HPV
antigen.

26. The method of claim 25, wherein the composition is administered in combination with pharmacologically acceptable components, such as, carrier, diluent or vehicle.

27. The method of 25, wherein the immune responses comprise a cell-mediated immune response.

28. A method of circumventing pre-existing immunity to authentic VLPs by using composition of claim 1 or claim 21 with different morphology compare to authentic VLPs to administer a host.

29. A method of circumventing existing immune tolerance to authentic VLPs by using composition of claim 1 or claim 21 with different morphology compare to authentic VLPs to administer a host.

30. A method of circumventing the problem associated with authentic VLPs of interference with commercial anti-capsid protein based assays by using composition of claim 1 or claims 21 with different morphology compare to authentic VLPs to administer a host.